WO2011106904A1 - Method for the assisted identification of inbred line of wuzhishan micropig and the specific primers thereof - Google Patents
Method for the assisted identification of inbred line of wuzhishan micropig and the specific primers thereof Download PDFInfo
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- WO2011106904A1 WO2011106904A1 PCT/CN2010/000252 CN2010000252W WO2011106904A1 WO 2011106904 A1 WO2011106904 A1 WO 2011106904A1 CN 2010000252 W CN2010000252 W CN 2010000252W WO 2011106904 A1 WO2011106904 A1 WO 2011106904A1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
Definitions
- the invention relates to a method for assisting identification of Wuzhishan miniature pig inbred line and a special primer thereof. Background technique
- Wuzhishan Mini-Pig WZSP
- the Wuzhishan miniature pigs were inbred by the siblings, their relatives, or their grandparents and their grandparents for more than 10 generations. The test proved that the genes were highly homozygous and the inheritance was very stable. It was named Wuzhishan miniature pig inbreds. (ZL02149030. 9).
- the inbred line Compared with the original breeding herd, the inbred line has better temperament, clear genetic background, high homogeneity of gene, black-and-white color, good consistency of body type and coat color, and smaller body size and genetic stability than domestic and foreign varieties.
- Advantages, belonging to the new animal genetic resources, has been listed as the National Livestock and Poultry Genetic Resources Conservation Plant (C1101001).
- human comparative medicine such as burn skin grafting, cardiovascular model, oral dental disease, xenotransplantation and other deep-level, multi-faceted research and development and utilization results also show that it is not only excellent for pig breeding, high-end meat processing. Materials are the best choice for experimental animal model research.
- the WZSP inbred line will play a special role and immeasurable contribution in the study of human comparative medical research and xenotransplantation.
- the invention provides a method for assisting identification of Wuzhishan miniature pig inbred line and/or non-Wuzhishan mini-pig inbred line, and detecting the sequence of the genomic DNA of the test pig from the 5' end of the genus K GenBank Access i on Number DQ406743 Is the 1273 deoxyribonucleotide G or A, and the genotype of the test pig is GG, GA or AA; the GG genotype is sequence 1 from the 5' end of the 1273 deoxyribonucleotide to G Homozygous; the AA genotype is a homozygous sequence 1 from the 5' end of the 1273 deoxyribonucleotide to A; the GA genotype is their hybrid;
- the pigs to be tested of the GG genotype are the candidate Wuzhishan miniature pig inbreds
- the pigs to be tested for the GA genotype and the pigs to be tested for the AA genotype were non-Wuzhishan minipig inbred lines. Determining the sequence 1 in the genomic DNA of the pig to be tested ( GenBank Access i on Number
- the method of DQ406743) from the 5' end of the 1273th deoxyribonucleotide to G or A includes the following steps: genomic DNA extraction, PCR amplification, restriction enzyme digestion and electrophoresis.
- the PCR-amplified primer pair satisfies the following conditions: using the pig's genome ⁇ as a template
- the PCR amplified product contains the sequence 1 ( GenBank Access i on Number DQ406743 )
- the PCR-amplified primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.
- the restriction endonuclease used for the digestion may specifically be 73 ⁇ 4 sl.
- the electrophoresis may specifically be agarose gel electrophoresis.
- Pigs with GG genotype were detected by PCR-RFLP (7ksl digestion) with four bands, 357 bp, 204 bp, 134 bp and 67 bp.
- AA genotype pigs were identified by PCR-RFLP (Tasl digestion). Electrophoresis bands with sequence lengths of 82 bp, 275 bp, and 204 bp, respectively.
- the present invention also protects a primer pair for assisting in the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line, the primer pair satisfying the following conditions:
- the PCR amplification product containing the genomic DNA of the pig as a template contains Deoxyribonucleotide at position 1273 from the 5' end of SEQ ID NO: 1.
- the primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.
- the primer pair can be applied to prepare a kit for assisting identification of Wuzhishan miniature pig inbred line and/or non-Mt. Wushan small pig inbred line.
- the invention also protects a kit for the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line containing the primer pair.
- the kit may further include a PCR reagent and an electrophoresis reagent.
- the kit or the primer pair can be applied to assist in the identification of Wuzhishan miniature pig inbred line and/or non-Wuxianshan minipig inbred line.
- the kit, the primer pair and the method are applicable to the breeding of pigs.
- the Wuzhishan miniature pig inbred line may specifically be the F 13th generation to the F 2 . generation.
- Figure 1 shows the restriction enzyme digestion of three genotypes; M: DNA molecular weight standard (100-1500bp ladder).
- the pigs used in the trial were as follows: 42 Guizhou mini-pigs, 49 Guangxi Bama miniature pigs and 42 heads Wuzhishan miniature pig inbred line.
- a pair of primers were designed according to the second intron sequence of the Pnas-4 gene (GenBank Accession Number DQ406743) (sequence 1 of the Sequence Listing) as follows:
- PCR reaction system 2.0 ⁇ L 10 times reaction buffer, 1.6 ⁇ LMgCl 2 (2.5 mmol/L), 1 ⁇ L upstream (1 ⁇ mol/L), 1 ⁇ L downstream (10 ⁇ mol/ L), 0.4 ⁇ L dNTPs (10 mmol/L), 0.2 ⁇ L Taq S
- PCR amplification procedure 95. C 3 min, 30 cycles (94 C 20 s, 62.5 ° C 30 s, 72 ° C 30 s), and finally extended at 72 ° C for 3 min.
- the amplified product was identified by 1.5% agarose gel electrophoresis.
- the enzyme digestion reaction system ( ⁇ : IX buffer 1 ⁇ L, PCR product 5wL, restriction endonuclease 7ksl 0.5 ⁇ L (5U), supplemented with 0 L of 10 L.
- the digested products showed three bands. Some samples showed four bands of electrophoresis with sequence lengths of 357 bp, 204 bp, 134 bp and 67 bp. Some samples showed five bands of electrophoresis, with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively. The remaining samples showed six The electrophoresis bands were 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
- PCR amplification products showing samples of different electrophoresis bands were recovered and purified by agarose gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), and the recovered DNA fragment was ligated to pGEM-T ( After Promega, according to the method of Cohen et al. ⁇ Proc Natl Acad Sci, 69:2110), the ligated product was transformed into E. coli DH5 ⁇ (Beijing Tianshi Times) competent cells according to the carboxy-penicillin resistance marker on the carrier. Positive clones were screened to obtain a recombinant plasmid containing the recovered fragment.
- this deoxyribonucleotide is GenBank Accession Number DQ406743 from the 5' end of the 1273 Deoxyribonucleotide, designated G1273A. Among them, the 1273 deoxyribonucleotide is
- the nucleotide sequence of the amplified fragment of G is shown in SEQ ID NO:1 of the Sequence Listing.
- genotypes were defined as follows:
- the homozygous genotype is GG
- PCR-RFLP detection (7ksl digestion) is four electrophoresis bands, and the sequence lengths are 357 bp, 204 bp, 134 bp and 67 bp, respectively;
- the allele is A
- its homozygous genotype is AA
- PCR-RFLP ( 7ksl digestion) is detected as five electrophoresis bands with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
- the heterozygous genotype of this locus was GA, and the PCR-RFLP (Tasl digestion) detected six electrophoresis bands with sequence lengths of 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
- the genomic DNA of the pig to be tested is PCR-amplified using the primer pair of the present invention to obtain a 762 bp fragment.
- the restriction fragment endonuclease Tasl was digested and the 357 bp fragment was divided into 82 bp and 275 bp fragments.
- the present invention finds a polymorphic locus.
- the Wuzhishan minipig inbred line only has the G allele at the polymorphic locus, while in other breeds of pigs, the A allele exists, so the polymorphic locus can be used.
- Screening for pigs to be tested If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan minipig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred.
- the method of the invention can be applied to the breeding of Wuzhishan miniature pig inbred line.
- Pig ear skin tissue samples were taken from the Wuzhishan Small Pig Inbred Line of the Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, including 15 F 13 generations, 13 F 14 generations, 11 F 15 generations, and 14 F 16 generations. 14 F 17 generations, 14 F 18 generations, 15 F 19 generations, F 2 . All samples from the 15th generation were treated with 75% alcohol and stored in a -2CTC refrigerator to prepare DNA.
- the primers are as follows:
- the amplified product was identified by 1.5% agarose gel electrophoresis.
- the enzyme digestion reaction system (lO L) is as follows:
- the Wuzhishan minipig inbred line is homozygous at the G1273A polymorphic locus, only the GG genotype.
- this polymorphic locus can be used to screen pigs for testing. If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan mini-pig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred.
- the method of the invention can be applied to the breeding of the Wuzhishan mini-pig inbred line, and all the pigs in the test pig group are firstly screened, the non-Wuzhishan mini-pig inbred line is eliminated, and the candidate Wuzhishan mini-pig inbred line is found, and the combination is obtained. Other methods are further confirmed.
- the invention can also be applied to detecting whether a commercially available Wuzhishan miniature pig inbred line is counterfeit.
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Abstract
The present invention provides a method for the assisted identification of inbred line of Wuzhishan micropig and the specific primers thereof. The method comprises detecting deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 of the pig to be detected that is G or A, and determining gene type of the pig to be detected that is GG, GA or AA; the GG gene type is a homozygote in which deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 is G; the AA gene type is a homozygote in which deoxyribonucleotide of the 1273 site of GenBank Accession Number DQ406743 is A; the GA gene type is a heterozygote of G and A; the pig to be detected of GG gene type is potential inbred line of Wuzhishan micropig; and the pig to be detected of GA gene type and AA gene type is the inbred line of non-Wuzhishan micropig. The method is useful for the breeding of inbred line of Wuzhishan micropig.
Description
辅助鉴定五指山小型猪近交系的方法及其专用引物 技术领域 Method for assisting identification of Wuzhishan miniature pig inbred line and its special primers
本发明涉及一种辅助鉴定五指山小型猪近交系的方法及其专用引物。 背景技术 The invention relates to a method for assisting identification of Wuzhishan miniature pig inbred line and a special primer thereof. Background technique
我国改革开放初期, 1983-1984年研究者在西德学习时, 有幸参观西 德哥廷根小型猪 (Gottengen Minieture Pig ) 育种中心, 了解到西方国 家已将小型猪大量应用于人类疾病动物模型, 同时又是猪胚胎工程极好的 研究对象, 哥廷根小型猪已销售到多个国家和地区, 于是产生了引回中国 开发应用的念头。 西德哥廷根小型猪育种专家之一施密特教授对中国梅山 猪感兴趣, 建议西德农业部列为双边合作研究专项支持, 研究者回国后也 得到农业部的资助。 但由于种种原因未能实现, 关键是西德不轻易通过合 作研究的方式把资源送给中国。 在此期间, 国内经历了大规模的家畜品种 资源调査, 发现了众多特异性畜禽品种, WZSP 就是其中一例, 但已濒临 灭绝 (见 1987年 7月原中国农业科学院畜牧研究所冯维奇研究员、 原广 东省畜牧研究所王正研究员等十七人参加的考察报告) 。 从资料上分析, WZSP的小型化特征、遗传稳定性要远远好于西德哥廷根小型猪, 将会具有 较好的应用前景。 In the early days of China's reform and opening up, when studying in West Germany from 1983 to 1984, researchers were fortunate enough to visit the Gottengen Minieture Pig Breeding Center in West Germany. It was learned that Western countries have applied large numbers of miniature pigs to animal models of human diseases. At the same time, it is an excellent research object for pig embryo engineering. Göttingen mini-pigs have been sold to many countries and regions, which has led to the idea of introducing China into development and application. Professor Schmidt, one of the small pig breeding experts in West Göttingen, is interested in China Meishan pigs. It is recommended that the Ministry of Agriculture of West Germany be listed as a special support for bilateral cooperation research. The researchers also received funding from the Ministry of Agriculture after returning home. However, for various reasons, the key is that West Germany does not easily send resources to China through cooperative research. During this period, the country experienced a large-scale investigation of livestock breed resources and found many specific breeds of livestock and poultry. WZSP is one of them, but it is on the verge of extinction (see July 1987, former researcher of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Feng Weiqi, The investigation report of 17 people including the former researcher of Wang Zheng, Guangdong Provincial Animal Husbandry Research Institute). From the data analysis, WZSP's miniaturization characteristics and genetic stability are far better than the West German Göttingen mini-pigs, which will have a good application prospect.
中国农业科学院畜牧研究所自 1987年至今历经 20年的努力, 在农业 部、 科技部、 国家自然科学基金委等先后资助下, 累计先后投资 1500 万 元, 利用我国体型小、 濒临灭绝的珍稀品种五指山小型猪 (WZSP) , 分离、 培育出世界上首批近交系猪, 目前已获得第 20代群体 (F2。) 。 将五指山 小型猪进行全同胞、 亲仔或祖孙三世代内近交繁育 10 代以上得到的近交 小型猪种群, 试验证明基因高度纯合、 遗传非常稳定, 被命名为五指山小 型猪近交系 (ZL02149030. 9 ) 。 培育的该近交系较之原始种猪群具有性情 温顺、 遗传背景清楚、 基因高度纯合、 体色为黑白花、 体型和毛色一致性 好, 较之国内外品种更具有体型小、 遗传稳定等优势, 属于新的动物遗传 资源, 已列为国家畜禽遗传资源保种场 (C1101001 ) 。 在人类比较医学, 如烧伤植皮、 心血管模型、 口腔牙齿疾病、 异种器官移植等深层次、 多方 位研究和开发利用结果亦表明: 它不仅是肉猪育种、 高档肉食加工的优良
素材, 更是实验动物模型研究的最佳选择。 WZSP近交系在人类比较医学研 究、 异种器官移植研究中将发挥特殊的作用和不可估量的贡献。 Since 1987, the Institute of Animal Science of the Chinese Academy of Agricultural Sciences has worked for 20 years. Under the assistance of the Ministry of Agriculture, the Ministry of Science and Technology, and the National Natural Science Foundation of China, it has invested a total of 15 million yuan to make use of rare and endangered species in China. Wuzhishan Mini-Pig (WZSP), isolated and cultivated the world's first inbred pigs, has now obtained the 20th generation group (F 2 .). The Wuzhishan miniature pigs were inbred by the siblings, their relatives, or their grandparents and their grandparents for more than 10 generations. The test proved that the genes were highly homozygous and the inheritance was very stable. It was named Wuzhishan miniature pig inbreds. (ZL02149030. 9). Compared with the original breeding herd, the inbred line has better temperament, clear genetic background, high homogeneity of gene, black-and-white color, good consistency of body type and coat color, and smaller body size and genetic stability than domestic and foreign varieties. Advantages, belonging to the new animal genetic resources, has been listed as the National Livestock and Poultry Genetic Resources Conservation Plant (C1101001). In human comparative medicine, such as burn skin grafting, cardiovascular model, oral dental disease, xenotransplantation and other deep-level, multi-faceted research and development and utilization results also show that it is not only excellent for pig breeding, high-end meat processing. Materials are the best choice for experimental animal model research. The WZSP inbred line will play a special role and immeasurable contribution in the study of human comparative medical research and xenotransplantation.
发明公开 Invention disclosure
本发明的目的是提供一种辅助鉴定五指山小型猪近交系的方法及其 专用引物。 It is an object of the present invention to provide a method for assisting in the identification of Wuzhishan miniature pig inbreds and its specific primers.
本发明提供了一种辅助鉴定五指山小型猪近交系和 /或非五指山小型 猪近交系的方法,是检测待测猪的基因组 DNA中序列 K GenBank Access i on Number DQ406743 ) 的自 5 '末端第 1273位脱氧核糖核苷酸是 G还是 A,确 定待测猪的基因型是 GG、 GA还是 AA; 所述 GG基因型为序列 1 的自 5 '末 端第 1273位脱氧核糖核苷酸为 G的纯合体; 所述 AA基因型为序列 1的自 5 '末端第 1273位脱氧核糖核苷酸为 A的纯合体; 所述 GA基因型为它们的 杂合体; The invention provides a method for assisting identification of Wuzhishan miniature pig inbred line and/or non-Wuzhishan mini-pig inbred line, and detecting the sequence of the genomic DNA of the test pig from the 5' end of the genus K GenBank Access i on Number DQ406743 Is the 1273 deoxyribonucleotide G or A, and the genotype of the test pig is GG, GA or AA; the GG genotype is sequence 1 from the 5' end of the 1273 deoxyribonucleotide to G Homozygous; the AA genotype is a homozygous sequence 1 from the 5' end of the 1273 deoxyribonucleotide to A; the GA genotype is their hybrid;
GG基因型的待测猪为候选的五指山小型猪近交系; The pigs to be tested of the GG genotype are the candidate Wuzhishan miniature pig inbreds;
GA基因型的待测猪和 AA基因型的待测猪为非五指山小型猪近交系。 所述确定待测猪的基因组 DNA中的序列 1 ( GenBank Access i on Number The pigs to be tested for the GA genotype and the pigs to be tested for the AA genotype were non-Wuzhishan minipig inbred lines. Determining the sequence 1 in the genomic DNA of the pig to be tested ( GenBank Access i on Number
DQ406743 ) 的自 5 '末端第 1273位脱氧核糖核苷酸是 G还是 A的方法包括 如下步骤: 基因组 DNA提取、 PCR扩增、 酶切和电泳。 The method of DQ406743) from the 5' end of the 1273th deoxyribonucleotide to G or A includes the following steps: genomic DNA extraction, PCR amplification, restriction enzyme digestion and electrophoresis.
所述 PCR扩增的引物对满足如下条件: 以猪的基因组匪为模板进行 The PCR-amplified primer pair satisfies the following conditions: using the pig's genome 匪 as a template
PCR扩增的产物含有序列 1 ( GenBank Access i on Number DQ406743 ) 的自The PCR amplified product contains the sequence 1 ( GenBank Access i on Number DQ406743 )
5 '末端第 1273位脱氧核糖核苷酸。 The 1273th deoxyribonucleotide at the 5' end.
所述 PCR扩增的引物对具体可为序列表的序列 2所示核苷酸和序列表 的序列 3所示核苷酸组成的引物对。 The PCR-amplified primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.
所述酶切所用的限制性内切酶具体可为 7¾sl。所述电泳具体可为琼脂 糖凝胶电泳。 GG基因型的猪, PCR-RFLP检测 (7ksl酶切) 为四条电泳条 带,序列长度分别为 357bp、204bp、134bp和 67bp ; AA基因型的猪, PCR-RFLP ( Tasl酶切)检测为五条电泳条带,序列长度分别为 82bp、 275bp、 204bp、 The restriction endonuclease used for the digestion may specifically be 73⁄4 sl. The electrophoresis may specifically be agarose gel electrophoresis. Pigs with GG genotype were detected by PCR-RFLP (7ksl digestion) with four bands, 357 bp, 204 bp, 134 bp and 67 bp. AA genotype pigs were identified by PCR-RFLP (Tasl digestion). Electrophoresis bands with sequence lengths of 82 bp, 275 bp, and 204 bp, respectively.
134bp和 67bp ; GA基因型的猪, PCR-RFLP ( Tasl酶切) 检测为六条电泳 条带, 序列长度分别为 357bp、 82bp、 275bp、 204bp、 134bp和 67bp ) 。
本发明还保护一种辅助鉴定五指山小型猪近交系和 /或非五指山小型 猪近交系的引物对, 所述引物对满足如下条件: 以猪的基因组 DNA为模板 进行 PCR扩增的产物含有序列 1的自 5 '末端第 1273位脱氧核糖核苷酸。 134bp and 67bp; GA genotype pigs, PCR-RFLP (Tasl digestion) detected six bands, the sequence length is 357bp, 82bp, 275bp, 204bp, 134bp and 67bp). The present invention also protects a primer pair for assisting in the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line, the primer pair satisfying the following conditions: The PCR amplification product containing the genomic DNA of the pig as a template contains Deoxyribonucleotide at position 1273 from the 5' end of SEQ ID NO: 1.
所述引物对具体可为序列表的序列 2所示核苷酸和序列表的序列 3所 示核苷酸组成的引物对。 The primer pair may specifically be a primer pair consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.
所述引物对可应用于制备辅助鉴定五指山小型猪近交系和 /或非五指 山小型猪近交系的试剂盒。 The primer pair can be applied to prepare a kit for assisting identification of Wuzhishan miniature pig inbred line and/or non-Mt. Wushan small pig inbred line.
本发明还保护一种含有所述引物对的辅助鉴定五指山小型猪近交系 和 /或非五指山小型猪近交系的试剂盒。 所述试剂盒还可包括 PCR试剂和 电泳试剂。 The invention also protects a kit for the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line containing the primer pair. The kit may further include a PCR reagent and an electrophoresis reagent.
所述试剂盒或所述引物对可应用于辅助鉴定五指山小型猪近交系和 / 或非五指山小型猪近交系。 The kit or the primer pair can be applied to assist in the identification of Wuzhishan miniature pig inbred line and/or non-Wuxianshan minipig inbred line.
所述试剂盒、 所述引物对和所述方法可应用于猪的育种中。 The kit, the primer pair and the method are applicable to the breeding of pigs.
所述试剂盒、 所述引物对、 所述方法和所述应用中, 所述五指山小型 猪近交系具体可为第 F13代至 F2。代。 In the kit, the primer pair, the method and the application, the Wuzhishan miniature pig inbred line may specifically be the F 13th generation to the F 2 . generation.
附图说明 DRAWINGS
图 1为三种基因型的样本的酶切鉴定图; M : DNA分子量标准 ( 100-1500bp ladder ) 。 Figure 1 shows the restriction enzyme digestion of three genotypes; M: DNA molecular weight standard (100-1500bp ladder).
实施发明的最佳方式 The best way to implement the invention
以下的实施例便于更好地理解本发明, 但并不限定本发明。 下述实施 例中的实验方法, 如无特殊说明, 均为常规方法。 下述实施例中所用的试 验材料, 如无特殊说明, 均为自常规生化试剂商店购买得到的。 The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
五指山小型猪近交系(李凯; 牟玉莲; 韩建林; 杨述林; 刘岚; 员新 旭; 郭勇; 冯书堂, Study on Genetic Variation of Inbred Fami l ies of Wuzhi shan Miniature Pig Us ing Mi crosatel l i te DNA Loc i , 畜牧兽医 学报 , Chinese Journal of Animal and Veterinary Sc i ences, 2009年 03期) 。 实施例 1、 Pnas-4基因 G1273A多态的发现 Wuzhishan Miniature Pig Inbred Lines (Li Kai; Yan Yulian; Han Jianlin; Yang Shulin; Liu Wei; Yuan Xinxu; Guo Yong; Feng Shutang, Study on Genetic Variation of Inbred Familys of Wuzhi shan Miniature Pig Us ing Mi crosatel li te DNA Loc i Journal of Animal Husbandry and Veterinary Medicine, Chinese Journal of Animal and Veterinary Sc ences, 2009, 03). Example 1. Discovery of Pnas-4 Gene G1273A Polymorphism
试验中用到的猪如下: 42头贵州小型猪、 49头广西巴马小型猪和 42头
五指山小型猪近交系。 The pigs used in the trial were as follows: 42 Guizhou mini-pigs, 49 Guangxi Bama miniature pigs and 42 heads Wuzhishan miniature pig inbred line.
1、 PCR扩增 1, PCR amplification
根据 Pnas- 4基因 (GenBank Accession Number DQ406743) (序列表 的序列 1) 的第二内含子序列设计一对引物如下: A pair of primers were designed according to the second intron sequence of the Pnas-4 gene (GenBank Accession Number DQ406743) (sequence 1 of the Sequence Listing) as follows:
上游引物(序列表的序列 2) : 5 ' - CTAGAACCACTCAAACCAAGCAGC- 3 ' ; 下游引物(序列表的序列 3) : 5 ' - ATCAGGCAGGTAAAAGGATAACGG- 3 ' 。 利用上述引物对, 以猪的基因组匪为模板进行 PCR扩增。 Upstream primer (sequence 2 of the sequence listing): 5 ' - CTAGAACCACTCAAACCAAGCAGC-3 '; downstream primer (sequence 3 of the sequence listing): 5 ' - ATCAGGCAGGTAAAAGGATAACGG-3'. Using the above primer pairs, PCR amplification was carried out using the pig's genome 匪 as a template.
PCR 反应体系:2.0μ L 10倍的反应缓冲液, 1.6μ LMgCl2(2.5mmol/L), 1 μ L上游弓 I物 (10μ mol/L) , 1 μ L下游弓 I物(10μ mol/L) , 0.4μ L dNTPs (lOmmol/L) , 0.2 μ L Taq S|, lwL模板, dd 0定容至 20 μ L。 PCR reaction system: 2.0 μL 10 times reaction buffer, 1.6 μ LMgCl 2 (2.5 mmol/L), 1 μL upstream (1 μmol/L), 1 μL downstream (10 μmol/ L), 0.4 μL dNTPs (10 mmol/L), 0.2 μL Taq S|, lwL template, dd 0 to 20 μL.
PCR扩增程序: 95。C 3min, 30个循环 (94。C 20s, 62.5°C 30s, 72°C 30 s) , 最后在 72°C延伸 3min。 PCR amplification procedure: 95. C 3 min, 30 cycles (94 C 20 s, 62.5 ° C 30 s, 72 ° C 30 s), and finally extended at 72 ° C for 3 min.
扩增产物经 1.5%琼脂凝胶电泳鉴定。 The amplified product was identified by 1.5% agarose gel electrophoresis.
2、 RFLP分析 2, RFLP analysis
酶切反应体系 (ΙΟμ : IX buffer 1 μ L, PCR产物 5wL, 限制性 内切酶 7ksl 0.5μ L (5U), 用 0补足 10 L。 The enzyme digestion reaction system (ΙΟμ: IX buffer 1 μL, PCR product 5wL, restriction endonuclease 7ksl 0.5μL (5U), supplemented with 0 L of 10 L.
酶切 8小时后, 用 1.5%琼脂糖凝胶电泳检测酶切结果。 After 8 hours of digestion, the digestion results were examined by 1.5% agarose gel electrophoresis.
酶切产物呈现 3种带型。 部分样本显示四条电泳条带, 序列长度分别 为 357bp、 204bp、 134bp和 67bp; 部分样本显示为五条电泳条带, 序列长 度分别为 82bp、 275bp、 204bp、 134bp和 67bp; 剩下的样本显示为六条电 泳条带, 序列长度分别为 357bp、 82bp、 275bp、 204bp、 134bp和 67bp。 The digested products showed three bands. Some samples showed four bands of electrophoresis with sequence lengths of 357 bp, 204 bp, 134 bp and 67 bp. Some samples showed five bands of electrophoresis, with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively. The remaining samples showed six The electrophoresis bands were 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
3、 克隆测序及序列分析 3. Cloning sequencing and sequence analysis
根据 PCR-RFLP分析结果, 分别将显示不同电泳条带的样本的 PCR扩 增产物用琼脂糖凝胶回收试剂盒 (天根生化科技有限公司) 回收纯化, 回 收的 DNA片段连接载体 pGEM-T(Promega 公司)后, 参照 Cohen等的方法 {Proc Natl Acad Sci, 69:2110) , 将连接产物转化大肠杆菌 DH5 α (北 京天为时代公司) 感受态细胞, 根据载体上的羧卞青霉素抗性标记筛选阳 性克隆, 得到含有回收片段的重组质粒。 以该重组质粒载体上的 Τ7和 SP6 启动子序列为引物对其进行核苷酸序列测定, 测序结果表明不同样本的扩 增片段长度均为 762bp, 只存在一个脱氧核糖核苷酸的差异 (G/A) , 此脱 氧核糖核苷酸为 GenBank Accession Number DQ406743的自 5'末端第 1273
位脱氧核糖核苷酸, 命名为 G1273A。 其中, 第 1273位脱氧核糖核苷酸为According to the PCR-RFLP analysis results, PCR amplification products showing samples of different electrophoresis bands were recovered and purified by agarose gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), and the recovered DNA fragment was ligated to pGEM-T ( After Promega, according to the method of Cohen et al. {Proc Natl Acad Sci, 69:2110), the ligated product was transformed into E. coli DH5 α (Beijing Tianshi Times) competent cells according to the carboxy-penicillin resistance marker on the carrier. Positive clones were screened to obtain a recombinant plasmid containing the recovered fragment. The nucleotide sequences of the Τ7 and SP6 promoter sequences on the recombinant plasmid were used as primers. The sequencing results showed that the amplified fragments of different samples were 762 bp in length, and there was only one difference in deoxyribonucleotides. /A) , this deoxyribonucleotide is GenBank Accession Number DQ406743 from the 5' end of the 1273 Deoxyribonucleotide, designated G1273A. Among them, the 1273 deoxyribonucleotide is
G的扩增片段的核苷酸序列如序列表的序列 1所示。 The nucleotide sequence of the amplified fragment of G is shown in SEQ ID NO:1 of the Sequence Listing.
根据测序结果和 PCR-RFLP结果, 对基因型进行如下限定: Based on the sequencing results and PCR-RFLP results, the genotypes were defined as follows:
若该位点的等位基因为 G,其纯合体基因型为 GG,PCR-RFLP检测(7ksl 酶切) 为四条电泳条带, 序列长度分别为 357bp、 204bp、 134bp和 67bp ; 若该位点的等位基因为 A, 其纯合体基因型为 AA, PCR-RFLP ( 7ksl酶 切) 检测为五条电泳条带, 序列长度分别为 82bp、 275bp、 204bp、 134bp 和 67bp ; If the allele of this locus is G, the homozygous genotype is GG, PCR-RFLP detection (7ksl digestion) is four electrophoresis bands, and the sequence lengths are 357 bp, 204 bp, 134 bp and 67 bp, respectively; The allele is A, its homozygous genotype is AA, and PCR-RFLP ( 7ksl digestion) is detected as five electrophoresis bands with sequence lengths of 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
该位点杂合体基因型为 GA, PCR-RFLP ( Tasl 酶切) 检测为六条电泳 条带, 序列长度分别为 357bp、 82bp、 275bp、 204bp、 134bp和 67bp。 The heterozygous genotype of this locus was GA, and the PCR-RFLP (Tasl digestion) detected six electrophoresis bands with sequence lengths of 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp, respectively.
采用本发明的引物对 PCR扩增待测猪的基因组 DNA, 得到 762bp片段。 待测猪的 GenBank Access ion Number DQ406743的自 5 '末端第 1273位脱氧 核糖核苷酸为 A时, 限制性内切酶 Tasl酶切后, 357bp的片段被分成 82bp 和 275bp的片段。 The genomic DNA of the pig to be tested is PCR-amplified using the primer pair of the present invention to obtain a 762 bp fragment. When the deoxyribonucleotide at position 1273 of the 5' end of GenBank Accession Number DQ406743 was A, the restriction fragment endonuclease Tasl was digested and the 357 bp fragment was divided into 82 bp and 275 bp fragments.
三种基因型的样本的酶切鉴定图见图 1。 The enzyme digestion identification of the samples of the three genotypes is shown in Figure 1.
不同品种的猪的基因型检测结果见表 1。 The genotype test results of different breeds of pigs are shown in Table 1.
表 1 不同品种猪的 Pnas-4基因 7ksl多态检测结果 Table 1 Pnas-4 gene 7ksl polymorphism test results of different breeds of pigs
结果表明, 五指山小型猪近交系只存在 GG基因型和 G等位基因, 而 其它品种的小型猪存在 A等位基因。 The results showed that there were only GG genotypes and G alleles in the Wuzhishan mini-pig inbred line, while the other alleles had the A allele.
本发明发现了一个多态位点, 五指山小型猪近交系在该多态位点只存 在 G等位基因, 而其它品种的猪, 存在 A等位基因, 所以该多态位点可以 用来对待测猪进行筛选。 如果待测猪为 GG基因型,那它可作为候选的五指 山小型猪近交系, 如果待测猪为 GA基因型或者 AA基因型, 那它肯定不是 五指山小型猪近交系。 本发明的方法可应用于五指山小型猪近交系的育 种。 可对待测猪群中的所有猪预先进行初步筛选, 淘汰非五指山小型猪近 交系, 找出候选的五指山小型猪近交系, 结合其它方法进行进一步确认。
本发明还可应用于检测市购的五指山小型猪近交系是否为假冒的。 实施例 2、 Pnas-4基因 G1273A多态的应用 The present invention finds a polymorphic locus. The Wuzhishan minipig inbred line only has the G allele at the polymorphic locus, while in other breeds of pigs, the A allele exists, so the polymorphic locus can be used. Screening for pigs to be tested. If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan minipig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred. The method of the invention can be applied to the breeding of Wuzhishan miniature pig inbred line. All pigs in the tested pigs were pre-screened in advance, the non-Wuzhishan mini-pig inbreds were eliminated, and the candidate Wuzhishan mini-pig inbreds were identified and further confirmed by other methods. The invention can also be applied to detecting whether a commercially available Wuzhishan miniature pig inbred line is counterfeit. Example 2, Application of Pnas-4 Gene G1273A Polymorphism
猪的耳皮肤组织样本均采自中国农业科学院北京畜牧兽医研究所的 五指山小型猪近交系, 其中 F13世代 15头, F14世代 13头, F15世代 11头, F16 世代 14头, F17世代 14头, F18世代 14头, F19世代 15头, F2。世代 15头全部样品 用 75%酒精处理后, 于 -2CTC冰箱保存, 备提 DNA。 Pig ear skin tissue samples were taken from the Wuzhishan Small Pig Inbred Line of the Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, including 15 F 13 generations, 13 F 14 generations, 11 F 15 generations, and 14 F 16 generations. 14 F 17 generations, 14 F 18 generations, 15 F 19 generations, F 2 . All samples from the 15th generation were treated with 75% alcohol and stored in a -2CTC refrigerator to prepare DNA.
1、 PCR扩增 1, PCR amplification
引物如下: The primers are as follows:
上游引物(序列表的序列 2) : 5 ' - CTAGAACCACTCAAACCAAGCAGC- 3 ' ; 下游引物(序列表的序列 3) : 5 ' - ATCAGGCAGGTAAAAGGATAACGG- 3 ' 。 利用上述引物对, 以猪的基因组匪为模板进行 PCR扩增。 Upstream primer (sequence 2 of the sequence listing): 5 ' - CTAGAACCACTCAAACCAAGCAGC-3 '; downstream primer (sequence 3 of the sequence listing): 5 ' - ATCAGGCAGGTAAAAGGATAACGG-3'. Using the above primer pairs, PCR amplification was carried out using the pig's genome 匪 as a template.
PCR 反应体系:2. Ομ L 10倍的反应缓冲液, 1.6 μ LMgCl2 (2.5mmol/L)PCR reaction system: 2. Ομ L 10 times reaction buffer, 1.6 μ LMgCl 2 (2.5mmol/L)
1 μ L上游弓 I物 (10μ mol/L) , 1 μ L下游弓 I物(10μ mol/L), 0.4 μ LdNTPs (10mmol/L) , 0.2 μ L Taq S|, lwL模板, dd 0定容至 20 μ L。 1 μL upstream I (10 μmol/L), 1 μL downstream (10 μmol/L), 0.4 μL dNTPs (10 mmol/L), 0.2 μL Taq S|, lwL template, dd 0 Capacitance to 20 μL.
PCR扩增程序: 95。C 3min, 30个循环 (94。C 20s, 62.5°C 30s, 72°C PCR amplification procedure: 95. C 3min, 30 cycles (94. C 20s, 62.5°C 30s, 72°C
30 s) , 最后在 72°C延伸 3min。 30 s), and finally extended at 72 ° C for 3 min.
扩增产物经 1.5%琼脂凝胶电泳鉴定。 The amplified product was identified by 1.5% agarose gel electrophoresis.
2、 RFLP分析 2, RFLP analysis
酶切反应体系 (lO L) 如下: The enzyme digestion reaction system (lO L) is as follows:
IX buffer 1 μ L, PCR产物 5 L, 限制性内切酶 Tasl 0.5 μ L (5U) , 用 0补足 10wL。 IX buffer 1 μL, PCR product 5 L, restriction endonuclease Tasl 0.5 μL (5U), supplemented with 0w 10μL.
酶切 8小时后, 用 1.5%琼脂糖凝胶电泳检测酶切结果, 并通过凝胶成 像系统获得最佳效果图片, 记录基因型。 After 8 hours of digestion, the results of the digestion were detected by 1.5% agarose gel electrophoresis, and the best effect picture was obtained by the gel imaging system, and the genotype was recorded.
结果见表 2。 The results are shown in Table 2.
表 2 不同世代的五指山小型猪近交系的基因型检测结果 Table 2 Genotypic test results of Wuzhishan mini-pig inbred lines from different generations
F16 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000F 16 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000
F17 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000F 17 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000
F18 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000F 18 14 14 0 0 1. 000 0. 000 0. 000 1. 000 0. 000
F19 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000F 19 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000
F2o 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000 结果表明, F13至 F2。代中, 均只有 GG基因型。 F 2 o 15 15 0 0 1. 000 0. 000 0. 000 1. 000 0. 000 The result shows that F 13 to F 2 . In the generation, only the GG genotype is available.
工业应用 Industrial application
本发明发现了五指山小型猪近交系在 G1273A 多态位点上是纯合的, 只有 GG基因型。 而其它品种的小型猪, 存在 GA、 AA基因型, 所以该多态 位点可以用来对待测猪进行筛选。 如果待测猪为 GG基因型,那它可作为候 选的五指山小型猪近交系, 如果待测猪为 GA基因型或者 AA基因型, 那它 肯定不是五指山小型猪近交系。 本发明的方法可应用于五指山小型猪近交 系的育种, 对待测猪群中的所有猪先进行初步筛选, 淘汰非五指山小型猪 近交系, 找出候选的五指山小型猪近交系, 结合其它方法进一步确认。 本 发明还可应用于检测市购的五指山小型猪近交系是否为假冒的。
The present inventors have found that the Wuzhishan minipig inbred line is homozygous at the G1273A polymorphic locus, only the GG genotype. For other breeds of minipigs, there are GA and AA genotypes, so this polymorphic locus can be used to screen pigs for testing. If the pig to be tested is the GG genotype, it can be used as a candidate for the Wuzhishan mini-pig inbred line. If the pig to be tested is a GA genotype or an AA genotype, it is certainly not a Wuzhishan minipig inbred. The method of the invention can be applied to the breeding of the Wuzhishan mini-pig inbred line, and all the pigs in the test pig group are firstly screened, the non-Wuzhishan mini-pig inbred line is eliminated, and the candidate Wuzhishan mini-pig inbred line is found, and the combination is obtained. Other methods are further confirmed. The invention can also be applied to detecting whether a commercially available Wuzhishan miniature pig inbred line is counterfeit.
Claims
1、 一种辅助鉴定五指山小型猪近交系和 /或非五指山小型猪近交系的 方法, 是检测待测猪的基因组匪中序列 1的自 5 '末端第 1273位脱氧核 糖核苷酸是 G还是 A,确定待测猪的基因型是 GG、 GA还是 AA; 所述 GG基 因型为序列 1的自 5 '末端第 1273位脱氧核糖核苷酸为 G的纯合体; 所述 AA基因型为序列 1的自 5 '末端第 1273位脱氧核糖核苷酸为 A的纯合体; 所述 GA基因型为它们的杂合体; GG基因型的待测猪为候选的五指山小型 猪近交系; GA基因型的待测猪和 AA基因型的待测猪为非五指山小型猪近 交系。 1. A method for assisting in the identification of Wuzhishan miniature pig inbreds and/or non-Wuzhishan minipig inbred lines, which is to detect the 1273 deoxyribonucleotides from the 5' end of the sequence 1 of the genome of the pig to be tested. G is A, determining whether the genotype of the test pig is GG, GA or AA; the GG genotype is a homozygosity of sequence 1 from the 5' end of the 1273 deoxyribonucleotide to G; the AA genotype The homozygote of the 1st position deoxyribonucleotide from the 5' end of the sequence 1 is A; the GA genotype is a hybrid thereof; the pig of the GG genotype is a candidate Wuzhishan minipig inbred; The pigs to be tested of the GA genotype and the pigs to be tested of the AA genotype were non-Wuzhishan minipig inbred lines.
2、 根据权利要求 1 所述的方法, 其特征在于: 所述检测待测猪的基 因组 DNA中序列 1的自 5 '末端第 1273位脱氧核糖核苷酸是 G还是 A的方 法包括如下步骤: 基因组 DNA提取、 PCR扩增、 酶切和电泳。 2. The method according to claim 1, wherein: the method for detecting whether the deoxyribonucleotide at position 1273 of the sequence 1 of the genomic DNA of the test pig is G or A from the 5' end comprises the following steps: Genomic DNA extraction, PCR amplification, restriction enzyme digestion and electrophoresis.
3、 根据权利要求 2所述的方法, 其特征在于: 所述 PCR扩增的引物 对满足如下条件: 以猪的基因组 DNA为模板进行 PCR扩增的产物含有猪的 基因组 DNA中序列 1的自 5 '末端第 1273位脱氧核糖核苷酸。 3. The method according to claim 2, wherein: the PCR-amplified primer pair satisfies the following conditions: PCR-amplified product using porcine genomic DNA as a template contains sequence 1 of porcine genomic DNA The 1273 deoxyribonucleotide at the 5' end.
4、 根据权利要求 3所述的方法, 其特征在于: 所述 PCR扩增的引物 对是序列表的序列 2所示核苷酸和序列表的序列 3所示核苷酸组成的引物 对。 The method according to claim 3, characterized in that the PCR-amplified primer pair is a primer set consisting of the nucleotide shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotide shown in SEQ ID NO: 3 of the Sequence Listing.
5、 根据权利要求 2至 4中任一所述的方法, 其特征在于: 所述酶切 所用的限制性内切酶为 7¾sl ; 所述电泳为琼脂糖凝胶电泳。 The method according to any one of claims 2 to 4, wherein the restriction endonuclease used for the digestion is 73⁄4 sl; and the electrophoresis is agarose gel electrophoresis.
6、 根据权利要求 1至 5 中任一所述的方法, 其特征在于: 所述五指 山小型猪近交系为第 F13至 F2。代。 6. The method according to any of claims 1 to 5, characterized in that: said Wuzhishan minipig inbred line for the first F 13 to F 2. generation.
7、 辅助鉴定五指山小型猪近交系和 /或非五指山小型猪近交系的引物 对, 所述引物对满足如下条件: 以猪的基因组 DNA为模板进行 PCR扩增的 产物含有序列 1的自 5 '末端第 1273位脱氧核糖核苷酸。 7. A primer pair for assisting the identification of the Wuzhishan minipig inbred line and/or the non-Wuzhishan minipig inbred line, the primer pair satisfying the following conditions: The PCR amplification product using the genomic DNA of the pig as a template contains the sequence 1 self. The 1273 deoxyribonucleotide at the 5' end.
8、 根据权利要求 7 所述的引物对, 其特征在于: 所述五指山小型猪 近交系为第 F13代至 F2。代。 8, according to claim 7, wherein the primer pair which is characterized in that: said Wuzhishan minipig inbred line for the first generation of F 13 to F 2. generation.
9、 序列表的序列 2所示核苷酸和序列表的序列 3所示核苷酸组成的 引物对。 9. A primer pair consisting of the nucleotides shown in SEQ ID NO: 2 of the Sequence Listing and the nucleotides shown in SEQ ID NO: 3 of the Sequence Listing.
10、 辅助鉴定五指山小型猪近交系和 /或非五指山小型猪近交系的试 剂盒, 含有权利要求 7至 9中任一所述的引物对。 10. A kit for assisting in the identification of a Wuzhishan minipig inbred line and/or a non-Wuzhishan minipig inbred line, comprising the primer pair of any one of claims 7-9.
11、 根据权利要求 10 所述的试剂盒, 其特征在于: 所述五指山小型 猪近交系为第 F13代至 F2。代。 11. The kit according to claim 10, wherein: said Wuzhishan minipig inbred line for the first generation of F 13 to F 2. generation.
12、 权利要求 7至 9中任一所述的引物对在制备辅助鉴定五指山小型 猪近交系和 /或非五指山小型猪近交系的试剂盒中的应用。 12. Use of a primer pair according to any one of claims 7 to 9 for the preparation of a kit for the auxiliary identification of Wuzhishan miniature pig inbreds and/or non-Wuzhishan minipig inbred lines.
13、 根据权利要求 12 所述的应用, 其特征在于: 所述五指山小型猪 近交系为第 F13至F2。代。 13. The use as claimed in claim 12, wherein: said Wuzhishan minipig inbred line for the first F 13 to F 2. generation.
14、 权利要求 Ί至 9中任一所述的引物对或权利要求 10或 11所述的 试剂盒在辅助鉴定五指山小型猪近交系和 /或非五指山小型猪近交系中的 应用。 The primer set according to any one of claims 9 to 10 or the kit according to claim 10 or 11 for assisting in the identification of Wuzhishan miniature pig inbred line and/or non-Wuzhishan minipig inbred line.
15、 根据权利要求 14 所述的应用, 其特征在于: 所述五指山小型猪 近交系为第 F13代至 F2。代。 15. The use of claim 14, wherein: said Wuzhishan minipig inbred line for the first generation of F 13 to F 2. generation.
16、 权利要求 1至 6中任一所述方法、 权利要求 Ί至 9中任一所述引 物对和权利要求 10或 1 1所述试剂盒中的任意一种在猪的育种中的应用。 Use of the method according to any one of claims 1 to 6, the primer set according to any one of claims 1 to 9 and the kit according to claim 10 or 1 1 in the breeding of pigs.
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US13/147,361 US9029090B2 (en) | 2010-03-01 | 2010-03-01 | Method for auxiliary identification of inbred line of wuzhishan miniature pig and its special primer |
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CN107306876A (en) * | 2017-05-31 | 2017-11-03 | 浙江甘源泓农业科技有限公司 | A kind of hybridization breezing and producing method of high-quality wild boar |
CN112094923A (en) * | 2020-09-30 | 2020-12-18 | 扬州大学 | SINE transposon polymorphic molecular marker associated with pig growth rate and detection method and application thereof |
CN112094923B (en) * | 2020-09-30 | 2022-07-12 | 扬州大学 | SINE transposon polymorphic molecular marker associated with pig growth speed as well as detection method and application thereof |
CN114574598A (en) * | 2022-04-02 | 2022-06-03 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying or assisting in identifying muscle area of costal eye of pig 5/6 |
CN114574598B (en) * | 2022-04-02 | 2023-08-08 | 中国农业科学院北京畜牧兽医研究所 | Method for identifying or assisting in identifying area of pig 5/6 rib eye muscle |
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